Astra 5 software

SEC-MALS experiments were performed using a fast protein liquid chromatography system connected to a Wyatt MiniDAWN TREOS instrument and a Wyatt Optilab rEX differential refractometer. Superdex 200 Increase 10/300 or Superose 6 Increase 10/300 gel filtration columns were pre-equilibrated with three different buffers (50 mM sodium acetate pH 4.5, 50 mM MES pH 6.0, or 50 mM Tris pH 8.0) in the presence of 100 mM NaCl and 1 mM TCEP normalized using ovalbumin and BSA. WT and D69A mutant PB1-ZZ proteins, prepared separately by the methods described earlier, were injected (1–3 mg/ml, 0.5 ml) at a flow rate of 0.5–0.75 ml/min. Data were analyzed using the Zimm model for static light scattering data fitting and represented using an EASI graph with a UV peak in the ASTRA V software (Wyatt).

SEC-MALS experiments were performed using a fast protein liquid chromatography system (GE Healthcare) connected to a Wyatt MiniDAWN TREOS MALS instrument and a Wyatt Optilab rEX differential refractometer. A Superdex 200 10/300 GL (GE Healthcare) gel-filtration column pre-equilibrated with equilibrium buffer [20 mM Tris-HCl (pH 8.0) and 300 mM NaCl] was normalized using ovalbumin protein. Proteins (2 mg) were injected at a flow rate of 0.5 ml/min. Data were analyzed using the Zimm model for static light-scattering data fitting and graphed using EASI graph with a UV peak in ASTRA V software (Wyatt Technology Corp., Goleta, CA, USA).

SEC-MALS experiments were performed using a fast protein liquid chromatography system (GE Healthcare) connected to a Wyatt MiniDAWN TREOS MALS instrument and Wyatt Optilab rEX differential refractometer. The column and buffer used were the same as those used in the final purification step. Ovalbumin was used as the isotropic scatterer for detector normalization. Light scattering from each sample (3–5 mg/ml, 0.5 ml) was measured and analysed using ASTRA V software (Wyatt).

All samples and buffers were filtered with a 0.1-μm pore size filter before use. ATP was added to each HK sample (∼15 μM, 400 μl) to a final concentration of 1 mM. Samples were then injected at a 0.4 ml/min flow rate onto a Superdex 200 GL 10/300 (or in the case of Fig. S4, Superdex 200 Increase GL 10/300) SEC column (GE Healthcare) using HSEC buffer with 1 mM ATP. For lit experiments, samples were illuminated with a blue LED panel for 1 min prior to injection, and the blue LED panel was faced toward the column during the run. For dark experiments, all steps were performed under dim red light. Samples were detected postelution by the inline miniDAWN TREOS light scattering and Optilab rEX refractive index detectors (Wyatt Technology). FPLC runs were performed at 4 °C, while MALS and refractive index measurements were at 25 °C. Data analysis and molecular weight calculations were performed using the ASTRA V software (Wyatt Technology). Raw data were plotted using RStudio. Assignments of oligomeric state are based on a qualitative, five-bin scale, with proteins considered to be “dimeric” or “monomeric” when their MALS-calculated mass falls within ±5% of the sequence-derived molecular weight for either state. Between these ranges are three bins of equal size: “mostly dimer,” “monomer/dimer mixture,” and “mostly monomer.”

SEC-MALS experiments were performed using an FPLC system (GE Healthcare) connected to a Wyatt MiniDAWN TREOS MALS instrument and Wyatt Optilab rEX differential refractometer (Santa Barbara, CA, USA). A Superdex 200 10/300 GL (GE Healthcare) gel-filtration column pre-equilibrated with buffer B was normalized using ovalbumin protein. Proteins were injected (3–10 mg/mL) at a flow rate of 0.4 mL/min. Data were analyzed using the Zimm model for fitting static light-scattering data and graphed using EASI graph with a UV peak in ASTRA V software (Wyatt).

Size-exclusion chromatography with multi-angle light scattering (SEC-MALS) experiments for XRCC6 (aa 1-609) and the FH domain of FOXL2 (aa 46-158) were performed using FPLC system (GE Healthcare) connected to a Wyatt MiniDAWN TREOS MALS instrument and a Wyatt Optilab rEX differential refractometer (Wyatt Technology, Santa Barbara, CA, USA). A Superdex 200 10/300 GL (GE Healthcare) gel filtration column pre-equilibrated with buffer A, was normalised using ovalbumin. Proteins were injected at a flow rate of 0.4 ml/min. Data were analysed using the Zimm model for static light-scattering data fitting, and graphs were constructed using EASI graph with a UV peak in the ASTRA V software (Wyatt Technology).